Phosphatidylserine-induced dissociation of the heterodimeric PstB2p/Pbi1p complex in yeast phosphatidylserine trafficking system.

Most phospholipids-essential building blocks of cellular membranes-are synthesized in the endoplasmic reticulum (ER) and distributed to the intracellular membranes. Yeast phosphatidylserine (PtdSer) is produced in the ER and is transported to the mitochondria, Golgi, or vacuoles; it is subsequently converted into phosphatidylethanolamine by phosphatidylserine decarboxylase. In yeast, PstB2p (Sec14p homolog) and Pbi1p are known to be involved in non-vesicular lipid transport from the ER to the Golgi, however, the molecular mechanisms remain unclear. In this study, we attempted to analyze the stoichiometric model of the PstB2p-Pbi1p complex in PtdSer transport from the ER to Golgi by using size exclusion chromatography with multi-angle static light scattering, mass spectrometry, reductive methylation, and homology modeling. The homology model of PstB2p was validated in part via reductive methylation, suggesting that it has structure similar to that of Sec14p. We observed that PstB2p forms a homodimer but exists as a 1:1 heterodimer in the presence of Pbi1p. When PtdSer was added to the PstB2p-Pbi1p complex, PtdSer bound to PstB2p, triggering the dissociation of the PstB2p-Pbi1p complex. PstB2p in complex with PtdSer exists as a monomer in contrast to its homodimeric form in the absence of PtdSer. These findings suggest that a stoichiometric model of the PstB2p-Pbi1p complex in yeast can be used to study the PtdSer transport system.

Structural insights into the apo-structure of Cpf1 protein from Francisella novicida.

Clustered regularly interspaced short palindromic repeats (CRISPRs) from Prevotella and Francisella 1 (Cpf1) are RNA-guided endonucleases that produce cohesive double-stranded breaks in DNA by specifically recognizing thymidine-rich protospacer-adjacent motif (PAM) sequences. Cpf1 is emerging as a powerful genome-editing tool. Despite previous structural studies on various Cpf1 proteins, the apo-structure of Cpf1 remains unknown. In the present study, we determined the solution structure of the Cpf1 protein from Francisella novicida (FnCpf1) with and without CRISPR RNA (crRNA) using small-angle X-ray scattering, providing the insights into the apo-structure of FnCpf1. The apo-structure of FnCpf1 was also visualized using negative staining electron microcopy. When we compared the apo-structure of FnCpf1 with crRNA-bound structure, their overall shapes (a closed form) were similar, suggesting that conformational change upon crRNA binding to FnCpf1 is not drastic, but a local induced fit might occur to recognize PAM sequences. In contrast, the apo Cpf1 from Moraxella bovoculi 237 (MbCpf1) was analyzed as an open form, implying that a large conformational change from an open to a closed form might be required for crRNA binding to MbCpf1. These results suggested that the crRNA-induced conformational changes in Cpf1 differ among species.

Design, synthesis and antiviral activity of 2-(3-amino-4-piperazinylphenyl)chromone derivatives.

Previously, we have confirmed that the antiviral activities of the chromone derivatives were controlled by the type as well as the position of the substituents attached to the chromone core structure. In the course of our ongoing efforts to optimize the antiviral activity of the chromone derivatives, we have been attempting to derivatize the chromone scaffold via introduction of various substituents. In this proof-of-concept study, we introduced a 3-amino-4-piperazinylphenyl functionality to the chromone scaffold and evaluated the antiviral activities of the resulting chromone derivatives. The synthesized 2-(3-amino-4-piperazinylphenyl)-chromones showed severe acute respiratory syndrome-corona virus (SARS-CoV)-specific antiviral activity. In particular, the 2-pyridinylpiperazinylphenyl substituents provided the resulting chromone derivatives with selective antiviral activity. Taken together, this result indicates the possible pharmacophoric role of the 2-pyridinylpiperazine functionality attached to the chromone scaffold, which warrants further in-depth structure-activity relationship study.

Synthesis and antiviral evaluation of 7-O-arylmethylquercetin derivatives against SARS-associated coronavirus (SCV) and hepatitis C virus (HCV).

Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduction of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydroxychromone pharmacophore which is in bioisosteric relationship with the 1,3-diketoacid moiety of the ADK. Thus, it was of our interest to test the antiviral activity of the quercetin derivatives with an arylmethyl group attached. In this study, we prepared a series of the 7-O-arylmethylquercetin derivatives with various aromatic substituents and evaluated their antiviral activity against the SARS-associated coronavirus (SARS-CoV, SCV) as well as hepatitis C virus (HCV). Single difference in the aromatic substituent fine-tuned the biological activity of the 7-O-arylmethylquercetin derivatives to result in two different classes of derivatives selectively active against SCV and HCV.

2,6-Bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis C virus (HCV) and SARS-associated coronavirus (SCV).

In this study, as a bioisosteric alternative scaffold of the antiviral aryl diketoacids (ADKs), we used 5-hydroxychromone on which two arylmethyloxy substituents were installed. The 5-hydroxychromones (5b-5g) thus prepared showed anti-HCV activity and, depending on the aromatic substituents on the 2-arylmethyloxy moiety, some of the derivatives (5b-5f) were also active against SCV. In addition, unlike the ADKs which showed selective inhibition against the helicase activity of the SCV NTPase/helicase, the 5-hydroxychromones (5b-5f) were active against both NTPase and helicase activities of the target enzyme. Among those, 3-iodobenzyloxy-substituted derivative 5e showed the most potent activity against HCV (EC(50) = 4 µM) as well as SCV (IC(50) = 4 µM for ATPase activity, 11 µM for helicase activity) and this might be used as a platform structure for future development of the multi-target or broad-spectrum antivirals.